This invention relates to the marking of materials and in particular to a method of marking a liquid and subsequently detecting that the liquid has been marked.
There is a widespread requirement to be able to trace the path taken by a given material as it moves from one location to another. In general terms, two broad categories of material movement are recognised:
(i) The movement of materials as a result of natural processes occurring in the biosphere, e.g., the flow of water in sub-surface aquifers, the movement of sediments etc.
(ii) The movement of materials which have been manufactured by man, i.e., items which do not occur in the natural environment or which are natural materials being transported as a result of man's activities. The former would include any item produced by man, and the latter items such as grain and other food materials, mineral ores and petroleum products, such as crude oil.
In all these situations, there may be reasons why it is necessary to develop specific procedures to trace these movements. It may be that direct observation is not possible, e.g., when following the path of an underground stream. It may be that it is necessary to monitor the movement of goods without the direct knowledge of the transporters or, for legal reasons, to prove that the appearance of a material at a particular point in the biosphere was due to the same material originating from a known starting point.
For example, it is undesirable and in certain circumstances illegal, for petroleum materials to leak from storage sites or transportation containers and contaminate the natural environment. Petrol storage tanks, e.g., at petrol filling stations, are usually located underground. Should one of these tanks develop a leak, the loss of material will eventually be detected, either by audits on the material being added to and removed from the storage tank, or by detection of spilt, leakage material at some site adjacent the storage tank area. Since the tanks are underground, visual inspection is not normally possible and it is a costly procedure to excavate successive tanks to determine which tank is the cause of the leakage. The normal procedure would be to develop a protocol whereby a known marker, e.g., a dye, is added to the tanks to determine, by tracing the movement of the dye, which tank is the cause of the leakage. Cheaper remedial action can then be taken to deal with the identified leaking tank. One feature of this procedure is that, in order to know which tank is leaking, the markers added to each tank must be different, i.e., if there are six tanks, then six different dyes, each recognisable by some property which can be accurately and uniquely determined, need to be used. The greater the number of individual components in a particular system, the greater the number of unique traces that need to be used to make the necessary distinction between the paths taken by different leaks from different tanks.
Another example concerns the identification of the source of pollution in the sea and waterways from spills of petroleum materials, particularly oil. The environmental damage caused by accidental oil spills and deliberate dumping of oil by ships, e.g., when washing tanks, is significant and there is a growing demand for the culprits to be identified and to be held responsible for clean-up operations. One of the problems associated with the identification of oil samples in large volumes of aqueous media, such as an oil slick on the sea, is that any marker introduced into oil has a tendency to partition out or be dispersed in the aqueous phase, rendering collection and identification of the marker particularly difficult.
A further example illustrating the need to monitor the movement of a liquid from one location to another is provided by the practice of adding to fuel oils, additives, such as antistatic agents, detergents etc., in order to improve the performance of the oil. It is important that persons dealing in such materials are aware whether they have been treated, but many of these additives are only added in amounts which cannot be detected without recourse to complex and often expensive analytical procedures, and in certain instances it is not readily possible to determine whether the additive has been added at all, because its presence is effectively masked by impurities in the fuel oil. For example, antistatic agents often incorporate chromium ions whose detection is relatively straightforward. However, naturally occurring levels of chromium in oil are often far in excess of that introduced by the antistatic agent. It has been proposed to dye the oil to indicate the presence of these additives, but the amount of dye which must be added to produce a visible colour change in such materials is unacceptable to both producer and consumer alike, e.g., for reasons of cost, possible loss of performance, potential damage to engines etc and the amount may exceed threshold limits set by standards.
Another example is provided by the exemption from value-added-tax (VAT) of fuel oils for agricultural machinery and seagoing vessels. It has been known for unscrupulous individuals to take advantage of this exemption, by using such fuel for purposes for which there is no exemption, such as motor cars, thereby depriving the government of revenue.
In addition, there are many reasons why individuals, corporations, public bodies and governments might wish to mark materials, e.g., to monitor the flow of materials along distribution and sales networks, in order to be able to determine the ultimate fate of that material and/or the efficiency of a particular distribution network compared with another.
Many tracing methods have been used to solve problems of this sort, all of which involve the addition of some characteristic marker, such as dyes or radioactive compounds, to the material being monitored. Biological materials, such as bacteriophage or bacteria have also been used, most notably for tracing the movement of water bodies in the natural environment. In these cases, the living systems possess some property (e.g., a known drug resistance pattern or particular host specificity) which does not normally occur in nature. The added organisms can be traced from their point of addition by obtaining samples as required, isolating any organisms in those samples, and showing that the organisms originally added can be isolated from the samples.
International Patent Publication No. WO 87/06383 discloses a method of labelling an item or substance which involves labelling with a macromolecule, such as nucleic acid or a polypeptide. The method takes advantage of the ability to detect the presence or absence of molecules, such as DNA or protein per se, by simple chemical analytical procedures, referred to as "YES/NO" tests, which indicate whether or not the macromolecule is present. For example, the presence of DNA can be detected by using non-specific chemical agents which bind to the DNA, such as ethidium bromide, acridine orange or bis-benzimide (H33258, Heochst dye 33258). In the case of ethidium bromide, this compound cannot be detected under normal visual light wavelengths. Labelling may therefore be achieved by providing DNA and ethidium bromide together. The presence of the DNA (with bound ethidium bromide) can subsequently be detected by ultraviolet irradiation. There is no discrimination between different DNA molecules from different sources, e.g., from different organisms.
The resolution of the system may, however, be considerably improved by taking advantage of the ability of macromolecules, such as nucleic acids and proteins, to be recognised unequivocally by a second complementary macromolecule to provide a unique marker. Accordingly, it is possible to determine the authenticity of an item or substance, by labelling that item or substance with a predetermined macromolecular first compound capable of binding to a second complementary macromolecular compound and using that second compound as a probe to determine the presence or absence of the first compound and thus establish whether a given item or substance is the genuine (marked) article.
The uniqueness of DNA to each species and, indeed, each strain within a species, together with the technical capacity to hybridise unique DNA molecules provides a more sophisticated form of labelling than a simple "YES/NO" test. For each strain of organism, the DNA (or RNA) molecules are unique, although different strains of the same species differ by virtue of small variations in sequences of bases. It is possible to recognise the DNA of different species and different strains of the same species by examining the DNA with labelled DNA probes. An item or substance may be labelled with a "signal DNA" comprising a sequence capable of hybridising with a specific "probe DNA". Both the signal DNA and the probe DNA are kept secret. Where analysis of the labelled item or substance by means of the probe DNA reveals the signal DNA, the item or substance is genuine. If not, the item or substance is an imitation.
This marking technique is primarily intended for labelling articles, such as luxury goods, e.g., watches, perfume and clothes; films and recordings; bank notes; art works; documents such as passports, and machinery and parts, e.g., for cars, although reference is made to labelling pharmaceuticals and other chemicals, such as fertilisers, herbicides and pesticides.
Labelling may be achieved in a variety of ways, e.g., the signal compound may be incorporated directly into the item or substance during its manufacture, or it may be attached by an adhesive. The signal compound may also be included in a material such as a paint or ink which is applied to an item or substance.
International Patent Publication No. WO 90/14441 discloses a method of monitoring the presence of a substance which comprises marking the substance with a nucleic acid tag, collecting the substance and detecting the tag, generally by amplifying the nucleic acid using polymerase chain reaction technology. The polymerase chain reaction (PCR) procedure is disclosed in, e.g., U.S. Pat. Nos. 4,683,202 and 4,683,195, and European Patent Publication Nos. 258017 and 237362, and allows for the enzymatic amplification, in vitro, of specific DNA sequences using oligonucleotide primers which recognise all or part of the DNA molecule used as the taggant. The use of PCR technology enables the DNA molecule to be amplified exponentially, e.g., 25 complete cycles of amplification enables a single DNA molecule to be increased 3.4.times.10.sup.7 times.
Also disclosed is a kit designed to tag and monitor substances comprising a nucleic acid taggant and a polynucleotide complementary to the taggant which can be either a signal probe, capture probe or a primer for the PCR method. Reference is made to the kits containing "signal means", such as enzymes, radio-isotopes and fluorescent labels, but no further details are provided.
Substances which may be tagged are said to include air pollutants, organic solvents (such as those from dry cleaners, chemical factories, airports and petrol filling stations), explosive compositions (such as plastic explosives and gunpowder), paper goods (such as newsprint, money and legal documents), pharmaceutical products (such as medicaments), inks, perfumes and paint products.
The nucleic acid may be free, i.e., naked, encapsulated by polymeric substances (such as proteins) or lipophilic compositions (such as liposomes), bound to a component of the tagged substance or bound to a solid support which is then mixed with the substance being tagged. Suitable support materials are said to include latex, dextran and magnetic beads, but no further details are provided.
Our copending International Patent Publication No. WO91/7265 also discloses a method for tracing the origin and movement of materials, both liquid and solid, which comprises: adding to the material a microtrace additive comprising DNA molecules; sampling the resulting material after movement thereof, and detecting the presence of the microtrace additive in the sample.
In a preferred aspect of the invention, the material being monitored is a liquid hydrocarbon, such as oil, and the microtrace additive is designed such that it cannot be easily removed from the hydrocarbon by aqueous washing, e.g., following an oil spill at sea. In mixtures of water and hydrocarbons, any DNA present in the hydrocarbon tends to move to the aqueous phase. The partitioning of DNA under these conditions is due to the negative charges associated with the phosphodiester groups of the DNA and the ability to form hydrogen bonds with water molecules and an inability to do so in a hydrocarbon environment. Various methods are proposed for ensuring that the DNA remains in the hydrocarbon rather than partitioning to any aqueous phase, including covalently linking the DNA to hydrophobic beads, typically of from 1 to 5 .mu.m diameter, designed to be soluble in hydrocarbons and not the aqueous phase.
By taking advantage of recent advances in techniques, such as PCR technology, for the detection of DNA at exceedingly low concentrations, only small quantities of DNA, typically in the concentration range 1.times.10.sup.-11 to 1.times.10.sup.-6 g DNA per ml of oil or other liquid, are used in the microtrace additive. For example, plasmid pBR322 DNA (2.times.10.sup.-9 g), chosen because DNA primers for amplification of this molecule are commercially available, was added to Arabian light crude oil (100 .mu.l) and mixed. To subsequently extract the DNA, distilled water (100 .mu.l) was added to the oil and the mixture thoroughly mixed to extract the pBR322 DNA from the oil into the aqueous phase. The oil-water mixture was centrifuged (10000 xg for 5 minutes) and the aqueous phase layer (5 .mu.l) removed and loaded into a standard Tag polymerase PCR reaction vial and reaction mixture (100 .mu.l containing KCl (50 mM); Tris-HCl buffer (10 mM; pH8.4); MgCl.sub.2 (1.5 mM); gelatin (100 .mu.g/ml); two pBR322 DNA primers (0.25 .mu.m); deoxyribose nucleotide phosphates (200 .mu.g of each of dATP, dCTP, dGTP, dTTP), and Tag polymerase (2.5 units). Following automated PCR cycling, the reaction mixture (10 .mu.l) was loaded onto agarose gel (2% w/v) and electrophoresed under standard conditions. The completed gel was stained with ethidium bromide to visualise the amplified DNA. No bands appeared in various negative controls.
Whilst DNA is particularly suitable for use as a unique marker, there are many instances where all that is required is a simple "YES/NO" test of the type described previously, e.g., to indicate that a particular fuel oil has been treated with a certain additive etc. In such circumstances, DNA is a less effective marker, as the DNA must either be present in prohibitively large amounts for it to be detected by non-specific assays, such as ethidium bromide staining, or PCR techniques are required to increase the amount of DNA to a level which can be detected. Thus, there is a continuing need for an accurate, reliable and cost-effective method of marking a liquid which is capable of providing a "YES/NO" test, and which does not rely on the use of complex, time-consuming analytical procedures or the use of unacceptably high levels of marker.
Many of the immunodiagnostic assays performed in clinical laboratories utilise a bioreactive molecule, typically an antibody, having a specific binding affinity for a target molecule, e.g., the antigen in respect of which the antibody was raised, in order to identify and/or isolate that target molecule in a given test sample. The bioreactive molecule is often coupled to the surface of a microbead, in order to increase the total surface area available to capture the target molecule and to facilitate the separation of bound target molecules from a solution of free molecules, since they can easily be immobilized, e.g., on a filter. Such beads are typically formed of a polymeric material, and generally have a diameter within the range from 0.05 to 100 .mu.m. The beads may be provided with a label, such as a fluorescent label, radiolabel etc., to provide signal means. Other beads are magnetic to aid their separation from the test sample, e.g., a magnet can be used to pull the beads into one region of the test vessel from which they can be physically separated. Magnetic beads can be prepared by dispersing particles of a magnetic material, such as magnetite (Fe.sub.3 O.sub.4), into the polymeric material used to form the particles.
Such microbeads are widely used in several fields of biochemistry and medicine, including the isolation of cells and target molecules from whole blood, tissue extracts, tissue cultures, enzyme digests and solid tissues; tissue typing; the isolation of PCR or Klenow DNA fragments; as carriers for pharmaceutical preparations; the separation of cancer cells from healthy cells; to provide a ready prepared template for genome walking, and the selective enrichment and/or isolation of pure and viable micro-organisms or smaller target compounds like soluble antigens, e.g., as disclosed in British Patent Publication No. 2017125, U.S. Pat. Nos. 4,035,316, 4,105,589, 4,138,383, 4,186,120, 4,224,198, 4,259,223, 4,267,237, 4,326,008, 4,369,226, 4,410,370, 4,510,244, 4,530,956, 4,550,017, 4,552,812, 4,563,510, 4,622,362, 4,654,267, 4,654,300, 4,663,277, 4,678,814, 4,689,307, 4,783,336, 4,828,984, 4,962,023, 5,028,545, and 5,081,020, and European Patent Publication Nos. 91453, 10986 and 106873.
Microbeads bearing fluorescent labels are commonly used to align, calibrate and correct apparatus, such as fluorescence microscopes and flow cytometers, e.g., as disclosed in U.S. Pat. Nos. 4,224,359, 4,714,682, 4,774,189, 4,857,451, 4,868,126, 4,918,004, 5,073,497, 5,084,394 and 5,093,234.